THROMBOSIS RESEARCH 68; 103-108,1992 00493848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
INCREASED PROCOAGULANT ACTIVITY IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH LIVER CIRRHOSIS
Yvon Calmus. *Annie Robert. Unite d’hepatogastroentkrologie and * Laboratoire central d’HCmatologie et d’lmmunologie, Hapital Saint-Antoine, 75012 Paris, France.
(Received 3.2.1992; accepted in revised form 10.8.1992 by Editor J. Soria)
ODUActivation of blood coagulation is a well recognized complication of liver cirrhosis (l-6), while a multifactorial pathogenesis has been presumed with contribution from reduced clearance of activated coagulation proteins (7), reduced synthesis of inhibitors (8,9), release of tissue thromboplastin from liver cells and systemic endotoxemia (10). Peripheral blood monocytes can initiate the coagulation protease pathway by several routes including tissue factor (1 l), monocyte prothrombinase (12) and factor VII/VIIa protease activity (13). Increased generation of monocyte procoagulant activity @‘CA)can be induced in vitro by a variety of stimuli (12,14,15). A number of studies suggest that monocyte PCA might play an important role in the activation of blood coagulation in vivo (16-21). The possible contribution of monocyte PCA to the coagulation activation in liver cirrhosis has never been considered. Therefore the aim of the present study was to evaluate the level of PCA expression in monocytes from cirrhotic patients and to determine whether there was any association between PCA and severity of cirrhosis. TS
AND
METHODS
Patients. Twenty seven patients with biopsy-proven liver cirrhosis (nineteen men, eight women; mean age 52 years, range 30-77) were studied. The etiology of cirrhosis was of alcoholic origin in twenty two patients, post-hepatitic in two patients and cryptogenic in the last three cases. Severity of cirrhosis was assessed by the Pugh modification of the Child-Turcotte (CT) classification (22) with the following variables : ascites, encephalopathy, serum albumin, serum bilirubin and prothrombin time. The best possible score of CT is 5 and the worst 15. Twenty healthy volunteers (thirteen men, seven women; mean age 51 years, range 35-71) served as controls. Mononuclear cells preparation. Peripheral blood mononuclear cells were isolated from heparinized venous blood, using aseptic technique. Platelet rich plasma was discarded, then mononuclear cell suspensions were prepared by centrifugation on lymphoprep@ (Nyegard, Oslo, Norway) at 1200 g for 12 min at 2O’C. The cell layer was collected at the interface and washed twice with RPM1 1640 (Flow Laboratories, Puteaux, France), containing 2 mM glutamine and 5
Key Words : Procoagulant activity, monocytes, cirrhosis. 103
104
PROCOAGULANTS
FROM BLOOD CELLS
Vol. 68, No. 1
p&ml gentamicin. The mononuclear cell suspensions contained less than 1 platelet per mononuclear cell, less than 1 % polymorphonuclear leukocytes and more than 95 % excluded trypan blue. Monocyte content was determined by non specific esterase staining (23). Mononuclear leukocytes were incubated at lx106/ml in serum free RPM1 1640 in 12x17 mm polypropylene tubes (Falcon, Grenoble, France) at 37’C, 5 % CO*, for 5 h. Cultures were performed either without stimuli or in the presence of LPS (Escherichia coli 011 l:B4, Sigma, St Louis, MI, USA) at a final concentration of 100 n&nl. At the end of incubation period, cells were centrifuged at 2000 g for 15 min and supematant was discarded; the cell pellets were washed twice in phosphate buffered saline and freezed at -70°C until clotting assay. Procoagulant activity assay. Cell lysates prepared by three cycles of freeze-thawing and sonication in Hepes saline (150 rnmol/L NaCl, 25 mmol/L Hepes) were assayed for PCA with a one stage clotting assay (24). A standard curve (log-log plot) derived from serial dilution of a rabbit brain thromboplastin (Simplastin @, Organon Teknica, Fresnes, France) was used to transpose procoagulant activity in arbitrary units. Clotting times for 0 and 1000 units were 200 and 45 s, respectively. Since PCA is generated exclusively by monocytes in this system (24,25), results were expressed as PCA per lo5 monocytes, taking into account the percentage of monocytes in the preparation of mononuclear cells. Endotoxin assay. All media and reagents were tested for endotoxin contamination by the Limulus amoebocyte lysate assay (Pyrogent, Mallinckrodt, St Louis, MO, USA), and contained no detectable endotoxin (~50 p&nl). Statistical methods. Data are expressed as the mean and standard derivation of triplicate assays. Significance of differences in mean laboratory parameters among the patient group and normal subjects was assessed using the two-tailed Student’s t test. Correlation coefficients were calculated using simple linear regression.
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CONTROL
L-
CIRRHOSlS P
BRIEF
COMMUNICATION
INCREASED PROCOAGULANT ACTIVITY IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH LIVER CIRRHOSIS
Yvon Calmus. *Annie Robert. Unite d’hepatogastroentkrologie and * Laboratoire central d’HCmatologie et d’lmmunologie, Hapital Saint-Antoine, 75012 Paris, France.
(Received 3.2.1992; accepted in revised form 10.8.1992 by Editor J. Soria)
ODUActivation of blood coagulation is a well recognized complication of liver cirrhosis (l-6), while a multifactorial pathogenesis has been presumed with contribution from reduced clearance of activated coagulation proteins (7), reduced synthesis of inhibitors (8,9), release of tissue thromboplastin from liver cells and systemic endotoxemia (10). Peripheral blood monocytes can initiate the coagulation protease pathway by several routes including tissue factor (1 l), monocyte prothrombinase (12) and factor VII/VIIa protease activity (13). Increased generation of monocyte procoagulant activity @‘CA)can be induced in vitro by a variety of stimuli (12,14,15). A number of studies suggest that monocyte PCA might play an important role in the activation of blood coagulation in vivo (16-21). The possible contribution of monocyte PCA to the coagulation activation in liver cirrhosis has never been considered. Therefore the aim of the present study was to evaluate the level of PCA expression in monocytes from cirrhotic patients and to determine whether there was any association between PCA and severity of cirrhosis. TS
AND
METHODS
Patients. Twenty seven patients with biopsy-proven liver cirrhosis (nineteen men, eight women; mean age 52 years, range 30-77) were studied. The etiology of cirrhosis was of alcoholic origin in twenty two patients, post-hepatitic in two patients and cryptogenic in the last three cases. Severity of cirrhosis was assessed by the Pugh modification of the Child-Turcotte (CT) classification (22) with the following variables : ascites, encephalopathy, serum albumin, serum bilirubin and prothrombin time. The best possible score of CT is 5 and the worst 15. Twenty healthy volunteers (thirteen men, seven women; mean age 51 years, range 35-71) served as controls. Mononuclear cells preparation. Peripheral blood mononuclear cells were isolated from heparinized venous blood, using aseptic technique. Platelet rich plasma was discarded, then mononuclear cell suspensions were prepared by centrifugation on lymphoprep@ (Nyegard, Oslo, Norway) at 1200 g for 12 min at 2O’C. The cell layer was collected at the interface and washed twice with RPM1 1640 (Flow Laboratories, Puteaux, France), containing 2 mM glutamine and 5
Key Words : Procoagulant activity, monocytes, cirrhosis. 103
104
PROCOAGULANTS
FROM BLOOD CELLS
Vol. 68, No. 1
p&ml gentamicin. The mononuclear cell suspensions contained less than 1 platelet per mononuclear cell, less than 1 % polymorphonuclear leukocytes and more than 95 % excluded trypan blue. Monocyte content was determined by non specific esterase staining (23). Mononuclear leukocytes were incubated at lx106/ml in serum free RPM1 1640 in 12x17 mm polypropylene tubes (Falcon, Grenoble, France) at 37’C, 5 % CO*, for 5 h. Cultures were performed either without stimuli or in the presence of LPS (Escherichia coli 011 l:B4, Sigma, St Louis, MI, USA) at a final concentration of 100 n&nl. At the end of incubation period, cells were centrifuged at 2000 g for 15 min and supematant was discarded; the cell pellets were washed twice in phosphate buffered saline and freezed at -70°C until clotting assay. Procoagulant activity assay. Cell lysates prepared by three cycles of freeze-thawing and sonication in Hepes saline (150 rnmol/L NaCl, 25 mmol/L Hepes) were assayed for PCA with a one stage clotting assay (24). A standard curve (log-log plot) derived from serial dilution of a rabbit brain thromboplastin (Simplastin @, Organon Teknica, Fresnes, France) was used to transpose procoagulant activity in arbitrary units. Clotting times for 0 and 1000 units were 200 and 45 s, respectively. Since PCA is generated exclusively by monocytes in this system (24,25), results were expressed as PCA per lo5 monocytes, taking into account the percentage of monocytes in the preparation of mononuclear cells. Endotoxin assay. All media and reagents were tested for endotoxin contamination by the Limulus amoebocyte lysate assay (Pyrogent, Mallinckrodt, St Louis, MO, USA), and contained no detectable endotoxin (~50 p&nl). Statistical methods. Data are expressed as the mean and standard derivation of triplicate assays. Significance of differences in mean laboratory parameters among the patient group and normal subjects was assessed using the two-tailed Student’s t test. Correlation coefficients were calculated using simple linear regression.
.
:
[-I.-
[
7
:* i..
I’
..
: :. ::
:’ :: :
CONTROL
L-
CIRRHOSlS P
